Although a quarter of all genes encode membrane proteins and the vast majority of therapeutic drugs target membrane proteins, remarkably little is known about membrane protein structure and function. This knowledge gap has occurred largely because often poor behavior of membrane proteins in detergent solution that leads to many intractable technical problems. We recently discovered that single point mutations in a marginally stable membrane protein, diacylglycerol kinase, can dramatically improve its stability in detergent. Moreover, stabilizing mutations are not rare. The goal of the work in this proposal is to learn more about how mutations stabilize membrane proteins in detergent. This work could lead to both practical improvements in our ability to handle membrane proteins and also provide fundamental insight into membrane protein structure. Aim I. Identify and characterize additional stabilizing mutations in diacylglycerol kinase and bacteriorhodopsin. This aim will not only provide fodder for subsequent aims, but by identifying a large number of mutations, it may be possible to develop predictive rules regarding side-chain types and sequence positions that are likely to stabilize. Aim II. Solve structures of stable mutants. We will learn in atomic detail how many of the mutants stabilize, providing new insights into how membrane protein structures are held together. Aim III. Examine the mechanism of inactivation in detergent solution for diacylglycerol kinase, and bacteriorhodopsin. This work will lead to a better understanding of why certain mutations stabilize and may also aid future detergent improvements by highlighting weaknesses in existing detergents. Aim IV. Develop a general screen for stabilizing mutations. We have devised a strategy for stability mutant screening that does not depend on a rapid activity assay. This method could bring the benefits of high stability to essentially any membrane protein of interest.